title: PROBINGαIIbβ3: LIGAND INTERACTIONS BY DYNAMIC FORCE SPECTROSCOPY AND SURFACE PLASMON RESONANCE.
abstract: The interaction between platelet integrinαIIbβ3 and fibrin(ogen) plays a key role in blood clot formation and stability. Integrin antagonists, a class of pharmaceuticals used to prevent and treat cardiovascular disease, are designed to competitively interfere with this process. However, the energetics of the integrin-drug binding are not fully understood, potentially hampering further development of this class of pharmaceuticals. We integrated dynamic force spectroscopy (DFS) and surface plasmon resonance (SPR) to probe the energetics of complex formation betweenαIIbβ3 and cHarGD, a cyclic peptide integrin antagonist. Analysis ofαIIbβ3:cHarGD DFS rupture force data at pulling rates of 14 000 pN/s, 42 000 pN/s and 70 000 pN/s yieldedkoff= 0.02-0.09 s-1, a dissociation energy barrier [Formula: see text] = 22-29 kJ/mol, and a potential well widthx-1= 0.5-0.8 nm. SPR kinetic data yielded an association rate constantkon= 7 × 103L/mol-s and a dissociation rate constantkoff= 10-2s-1, followed by a slower stabilization step (τ~ 400 s). Both DFS and SPR detected minimal interactions betweenαIIbβ3 and cHarGA demonstrating a key role for electrostatic interactions between the ligand aspartate and the integrin metal ion-dependent adhesion site (MIDAS). Our work provides new insights into the energy landscape ofαIIbβ3's interactions with pharmacological and physiological ligands.
authors.affiliation: Department of Physics, Wake Forest University 1834 Wake Forest Road, Winston-Salem, NC 27106, USA.
authors.forename: Samrat
authors.initials: S
authors.lastname: Dutta
authors.affiliation: Department of Biochemistry Wake Forest University School of Medicine Medical Center Boulevard Winston-Salem, NC 27157-1016, USA.
authors.forename: David A
authors.initials: DA
authors.lastname: Horita
authors.affiliation: Department of Biochemistry Wake Forest University School of Medicine Medical Center Boulevard Winston-Salem, NC 27157-1016, USA.
authors.forename: Roy R
authors.initials: RR
authors.lastname: Hantgan
authors.affiliation: Department of Physics, Wake Forest University 1834 Wake Forest Road, Winston-Salem, NC 27106, USA.
authors.forename: Martin
authors.initials: M
authors.lastname: Guthold
keywords: Surface plasmon resonance; association/dissociation kinetics; atomic force microscopy; dynamic force spectroscopy; receptor: ligand binding